Many methods have been developed over the last 30 years to facilitate the introduction of nucleic acid into cells which have greatly assisted, inter alia, our understanding of the control of gene expression.
Conventional methods to introduce DNA into cells are well known in the art and typically involve the use of chemical reagents, cationic lipids or physical methods. Chemical methods which facilitate the uptake of DNA by cells include the use of DEAE-Dextran (Vaheri and Pagano, Science 175:434). DEAE-dextran associates with and introduces the DNA into cells. However this can result in loss of cell viability. Calcium phosphate is also a commonly used chemical agent which, when co-precipitated with DNA, introduces the DNA into cells (Graham et al Virology (1973) 52: 456).
The use of cationic lipids (e.g. liposomes (Felgner (1987) Proc.Natl.Acad.Sci USA, 84:7413) has become a common method since it does not have the degree of toxicity shown by the above described chemical methods. The cationic head of the lipid associates with the negatively charged nucleic acid backbone of the DNA to be introduced. The lipid/DNA complex associates with the cell membrane and fuses with the cell to introduce the associated DNA into the cell. Liposome mediated DNA transfer has several advantages over existing methods. For example, cells which are recalcitrant to traditional chemical methods are more easily transfected using liposome mediated transfer.
More recently still, physical methods to introduce DNA have become effective means to reproducibly transfect cells. Direct microinjection is one such method which can deliver DNA directly to the nucleus of a cell (Capecchi (1980) Cell, 22:p 479). This allows the analysis of single cell transfectants. So called “biolistic” methods physically insert DNA into cells and/or organelles using a high velocity particles coated with DNA (Neumann (1982) EMBO J, 1:841).
Electroporation is arguably the most popular method to transfect DNA. The method involves the use of a high voltage electrical charge to momentarily permeabilise cell membranes making them permeable to macromolecular complexes. However physical methods to introduce DNA do result in considerable loss of cell viability due to intracellular damage. These methods therefore require extensive optimisation and also require expensive equipment.
More recently still a method termed immunoporation has become a recognised techinque for the introduction of nucleic acid into cells, see Bildirici et al Nature (2000) 405, 298. The technique involves the use of beads coated with an antibody to a specific receptor. The transfection mixture includes nucleic acid, typically vector DNA, antibody coated beads and cells expressing a specific cell surface receptor. The coated beads bind the cell surface receptor and when a shear force is applied to the cells the beads are stripped from the cell surface. During bead removal a transient hole is created through which nucleic acid and/or other biological molecules can enter. Transfection efficiency of between 40–50% is achievable depending on the nucleic acid used.
Typically, gene therapy involves the transfer, and optionally the stable insertion, of new genetic information into cells for the therapeutic treatment of disease. Genes that have been successfully expressed in mice after transfer by retrovirus vectors include human hypoxanthine phosphoribosyl transferase (Miller A et al, 1984, Science 255:630). Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance genes.
Transformation of hematopoietic progenitor cells to drug resistance by eukaryotic virus vectors has also accomplished with recombinant retrovirus based vector systems (Hock R A and Miller A D 1986, Nature 320:275–277; Joyner, et al. (1983) Nature 305:556–558; Williams D A et al 1984, Nature 310:476–480; Dick J E et al, 1985, Cell 42:71–79); Keller G et al 1985, Nature 318: 149–154; Eglitis M et al, 1985, Science 230: 1395–1398). Adeno-associated virus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance (Hermonat P L and Muzyczka N, 1984, supra; Tratschin J D et al, 1985, Mol. Cell. Biol. 5:3251). Other viral vector systems that have been investigated for use in gene transfer include papovaviruses arid vaccinia viruses (See Cline, M L (1985) Pharmac. Ther. 29: 69–92).
The main issues with respect to gene therapy relate to the efficient targeting of nucleic acid to cells and the establishment of high level transgene expression in selected tissues. A number of methodologies have been developed which purport to facilitate either or both of these requirements. For example, U.S. Pat. No. 6,043,339 discloses the use of signal peptides which when fused to nucleic acid, can facilitate the translocation of the linked nucleic acid across cell membranes. U.S. Pat. No. 6,083,714 discloses a combined nucleic acid and targetting means which uses the polycation poly-lysine coupled to an integrin receptor thereby targetting cells expressing the integrin. EP1013770 discloses the use of nuclear localisation signals (NLS) coupled to oligonucleotides. The conjugate may be covalently linked to vector DNA and the complex used to transfect cells. The NLS sequence serves to facilitate the passage of the vector DNA across the nuclear membrane thereby targetting gene delivery to the nucleus.
Nucleic acid, for example vector DNA, may be introduced into an animal via a variety of routes including enterally (orally, rectally or sublingually) or parenterally (intravenously, subcutaneously, or by inhalation).
It is known that introduction of certain aqueous solutions into the peritoneal cavity can be useful in the treatment of patients suffering from renal failure. Such treatment is known as peritoneal dialysis. The solutions contain electrolytes similar to those present in plasma; they also contain an osmotic agent, normally dextrose, which is present in a concentration sufficient to create a desired degree of osmotic pressure across the peritoneal membrane. Under the influence of this osmotic pressure, an exchange takes place across the peritoneal membrane and results in withdrawal from the bloodstream of waste products, such as urea and creatinine, which have accumulated in the blood due to the lack of normal kidney function. While this exchange is taking place, there is also a net transfer of dextrose from the solution to the blood across the peritoneal membrane, which causes the osmolality of the solution to fall. Because of this, the initial osmolality of the solution must be made fairly high (by using a sufficiently high concentration of dextrose) in order that the solution continues to effect dialysis for a reasonable length of time before it has to be withdrawn and replaced by fresh solution.
Other osmotic agents have been proposed for use in peritoneal dialysis and in recent years dextrin (a starch hydrolysate polymer of glucose) has been used. When instilled in the peritoneal cavity, dextrin is slowly absorbed via the lymphatic system, eventually reaching the peripheral circulation. The structure of dextrin is such that amylases break the molecule down into oligosaccharides in the circulation. These are cleared by further metabolism into glucose.
Typically, a medium chosen to introduce gene therapy materials to a patient via a body cavity might be a buffered saline solution, for instance, phosphate buffered saline (PBS).
Dextrin solutions have been proposed as the medium for delivery of drugs to the body via the peritoneum. In GB-A-2207050, such a solution is proposed for the intraperitoneal administration of drugs for which enteral administration is unsatisfactory. Such an approach is stated to be particularly useful for the delivery of peptide drugs such as erythropoetin and growth hormones. Reference is also made to cephalosporin antibiotics. Dextrin solutions have also been described for the administration of chemotherapeutic agents in the treatment of ovarian cancers. The use of icodextrin formulations to increase the efficacy of chemotherapeutics (especially 5FU) by increasing their dwell time in the peritoneal space is well described in Dobbie J W. (1997) Adv Perit Dial. 13: 162–7 and McArdle C S, et al. (1994) Br J Cancer 70(4):762–6.
The present invention is directed to a dextrin containing solution which shows enhanced ability to deliver nucleic acid to cells resulting in high level expression of transfected genes.